Method for isolating DNA

ABSTRACT

The invention describes a method for the isolation of components from samples, particularly large molecular weight DNA from biological samples. The method involves the application of controlled oscillatory mechanical energy to the sample for short periods of time of about 5 to 60 seconds to lyse the sample and release the component(s) from the sample, followed by standard isolation methods. In preferred embodiments, the method includes the use of a spherical particle for applying the mechanical energy.

FIELD OF THE INVENTION

This invention relates to reagents, methods and apparatus for theisolation of cellular components such as deoxyribonucleic acid (DNA),ribonucleic acid (RNA), proteins and other materials from naturalcellular sources or other sources containing these materials.

BACKGROUND OF THE INVENTION

Cells contain a wide variety of cellular components appropriate to theirfunction. They contain, for example, DNA and their expression productsincluding a host of proteinaceous materials. This invention is usefulfor the isolation of such cellular components, but in particular, theinvention is principally suited for the isolation of nucleic acids, DNAand RNA.

DNA is a critical component in the sequence of biological reactionswhich results in the expression of the myriads of proteins includinghormones, enzymes and structural tissue essential for the existence ofall forms of life. There is a critical need for small and large amountsof DNA for research purposes as well as diagnostic and therapeutic uses.

Plant/animal cells, tissues and organs, insects and microorganismsincluding viruses, yeast, fungi, algae and bacteria, and other materialsare potential sources of DNA. However, the structural organization ofsome of these sources can be so strong such that it is difficult, timeconsuming and may require expensive equipment to isolate DNA from thosetissues.

For instance, DNA isolation from certain bacteria is difficult becausethe cell walls are not readily susceptible to lysis. Current protocolsfor isolating DNA from bacteria frequently employ enzymes such aslysostaphin or lysozyme to digest the bacterial cell wall followed bythe addition of denaturing agents to lyse cells and inactivate thenucleases.

BRIEF SUMMARY OF THE INVENTION

The isolation of nucleic acids from various sources, particularlyplants, yeast, bacteria, and certain tissues, such as muscle, bone,cartilage, seeds, bark and the like, is difficult due to the presence ofcellular structures which protect the tissue, such as rigid cell walls,or other rigid structures, and therefore difficult to rupture completelywith commonly used buffers. Removal of these obstacles is tedious andnot always feasible with available methods. Variations in nucleic acidyield and quality from the various extraction procedures probably arisesfrom the non-homogeneity (inconsistency) of the tissue as it is brokenup. Thus, there is a need for a new technique for disrupting the tissueby a thorough, yet delimited mechanism to allow the rapid isolation ofnucleic acids in a reproducible manner without the need to excessivelyhomogenize the cells or tissues.

Procedures have now been discovered which makes possible the separationand isolation of large molecular weight DNA of exceptionally highquality in high yields from a variety of tissues. These procedure arevery convenient and can be completed in a very short period of time,typically less than one half hour. This process is, moreover, applicablenot only to intact biological tissue but also to microorganisms such asbacteria and yeast, and also to plant tissues as sources of DNA. Suchsources, especially bacteria, yeast and plants are much more convenientthan complex biological tissue from higher organisms as a source of DNAbecause they are uniform, readily available in any desired quantitiesand easier to work with than biological tissue.

The novel procedure of this invention comprises the application ofsufficient mechanical energy to the cell walls of the selected DNAsource to disrupt the cell walls and release the DNA. The essence ofthis invention is the discovery of the present methods for tissue orcell disruption in which the tissues and/or cell walls are fractured byspecified forces created by the reciprocal motion producing themechanical energy in a container with the tissue and liquid medium,thereby releasing the DNA from the tissue and into the medium.

In some preferred embodiments, the method includes the use of tissueand/or cell wall fracturing particles in the disruptive media in aclosed container.

After lysis of the tissue, the released DNA can be recovered in highyield and purity by any of a variety of recovery methods. Exemplary DNArecovery methods are described further herein.

There are a number of advantages provided by the process of thisinvention especially when conducted for the isolation of DNA. Theseinclude:

1. Applicability to DNA sources such as bacterial cells, fungi, plantcells and other intractable sources which have heretofore beenrefractory to homogenization procedures with any other extractant mediaor manipulation.

2. Recovery of DNA as a high yield product substantially uncontaminatedby other cellular components.

3. Applicability to the production of both small and large quantities ofDNA in batch, multiple sample or continuous processes.

4. Completion in a very short period of time.

5. No ultracentrifugation is required.

6. Isolation of high molecular weight DNA.

7. The reagents used in the methods of the invention are substantiallynon-toxic, odor free and readily available at commercially attractiveprices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a vertical side elevational view of the apparatus used in theinvention as it is housed in a casing, a side wall of the casing beingremoved for convenience of depiction and some parts being shown insection, there being depicted several specimen containing vesselreceivers on the holder disc and showing further a tilting of the vesselholder in a position denoting the vertical extremes of the verticaloscillating movement to which it is subjected during apparatusoperation;

FIG. 2 is a fragmentary view of the FIG. 1 apparatus on enlarged scale;

FIG. 3 is a top plan view of FIG. 2 and illustrates a fingered lockingplate employed with the apparatus and having a lock member to lock thespecimen vessels securely on the vessel holder to prevent relativemovement between the vessels and the holder during oscillatory movementof the holder, the locking plate being in a clearing position asrequired for access to the holder receptor structure when mounting anddemounting vessels;

FIG. 4 is a view the same as FIG. 3 except the locking plate is shown ina circularly moved position wherein the fingers thereof superpose overthe tops of the vessels and apply force to hold the vessels againstmovement relative to the holder during oscillatory movement;

FIG. 5 is a fragmentary vertical sectional view of a peripheral portionof the vessel holder depicting another form of lock member for clampingthe locking plate tightly against the holder so that clamping force isexerted by the fingers against vessel tops;

FIG. 6 is a fragmentary elevational view of a portion of the vesselholder and an anchor structure showing halter means wherein magnets areemployed to halter the holder against rotation in unison with themounting collar during operation of the apparatus;

FIG. 7 is a fragmentary elevational view taken on the line VII-Vii inFIG. 6;

FIG. 8 is a fragmentary plan view of a peripheral portion of the vesselholder illustrating a further embodiment of halter means wherein a postand keeper ring are used, one of such elements being mounted on theanchor structure and the other on the vessel holder;

FIG. 9 is a fragmentary elevational view of the structure depicted inFIG. 8;

FIG. 10 is a vertical central sectional view on enlarged scale of aspecimen vessel specially suited for use with the apparatus of theinvention and which embodies a casing encircling the specimen holdingpart of the vessel, the casing holding a heat absorbing medium fordrawing heat from the specimen and vessel during oscillation of theapparatus; and

FIG. 11 illustrates in panels A-O various configurations of particlesand containers for use in the present methods.

FIG. 12 presents a photograph of sample containers A-D, illustrating theappearance of containers of disrupted tissue according to the methodsdescribed in Example 8.

FIG. 13 illustrates the results of agarose gel electrophoresis, wherelanes A-D correspond to samples A-D processed as described in Example 8.

FIG. 14 illustrates the results of agarose gel electrophoresis, whereLanes 1-7 contain a sample from tubes 1-7, respectively, and Lane Ccontains control lambda DNA digested with Hind III as molecular weightmarkers, prepared as described in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the separation of components in asample, and particularly for the isolation of nucleic acids such as DNAfrom a tissue. The method is based, primarily, on the discovery ofprocedures for disruption of a sample, tissue or cell by the applicationof large controlled mechanical energy to the sample in a short period oftime, thereby facilitating the separation of components in the disruptedsample.

In a preferred embodiment, the invention describes a method for theisolation of high molecular weight DNA from a tissue or other sample.However, although many of the descriptions recite DNA isolation asexemplary for the methods, these descriptions are made for convenienceand to avoid redundancies. Therefore, the method is not to be construedas limited to DNA isolation but rather to be read on the isolation ofany sample component where disruption facilitates isolation according tothe present methods.

The first step in the practice of this invention is to mechanicallyfracture the cell walls, subcellular organelles, and/or tissue orextra-tissue structure of the DNA source material by the application ofthe controlled mechanical energy in a liquid medium containing the DNAsource material. The DNA source material can be bacteria, eukaryoticcells of a biological tissue, plant, yeast or fungi cells, ornon-cellular material, such as processed or unprocessed food, gels, soilsample, industrial solutions, and the like materials.

The application of uncontrolled mechanical energy leads to efficienttissue homogenization, however, the isolated DNA is typically ofrelatively small size which is undesirable for a variety of uses. Thepresent methods for applying mechanical energy produce DNA of largemolecular weight, typically greater that 10 kilobases (kb) averagemolecular weight.

Following fracture of the DNA source material, thereby releasing the DNAfrom its structural confines in the source material, the DNA isrecovered. Recovery of DNA can be conducted by any of a variety ofmethods, although certain recovery procedures are preferred.

A. Mechanical Lysis of the Tissue Source

An important aspect of the mechanical energy involved in sample, tissueor cell disruption in accordance with this invention is that the energyis reciprocally applied in an oscillatory motion to a liquid mediumcontaining the sample, thereby exerting a force sufficient to disruptthe tissue structure sufficient to release the DNA from the samplematerial's organized structure, such as cellular organelles of a tissue,into the liquid medium.

In some preferred embodiments, the mechanical energy is exerted in thepresence of one or more particles which function to impact the tissue,mix the liquid medium, and otherwise assist the isolation process.

The applied mechanical energy is controlled by the presence/absence ofparticles of different sizes, shapes and densities, together with thechoice of oscillation conditions (speed, periodicity, acceleration,etc.). Different materials as DNA source require application ofdifferent mechanical energies for efficient homogenization of thematerial and release of large molecular weight DNA. Examples ofdifferent applied energy conditions for different materials are givenlater.

Rotational energy such as generated with a blender or other homogenizeris not useful because the fragments of tissue, or cells of the tissue,simply rotate and do not collide with each other or the components ofthe liquid medium under sufficient mechanical energy of an oscillatorynature to lyse the tissue components and release nucleic acids.

1. Apparatus for Applying Mechanical Energy

It is known in the art to mechanically lyse source material to releasegenetic material such as RNA or DNA. Generally this involves subjectingthe source material to mechanical force and energy that disrupts thecells with violent impact action with consequent release of the nucleicacids. The released DNA or RNA then is recovered, e.g., from a liquidphase of the starting material, such procedure being known in the art.

One mechanical lysing protocol previously described for isolating DNAemploys bead mill separation, this source material being confined in avessel in a liquid phase thereof, there also being minute or small sizedbeads contained in the vessel. Rapid oscillation of the vessel is usedto impart impact energy to the beads and these strike the sourcematerial cells repeatedly to open the cells so the nucleic acids can bereleased.

Certain known separation devices and particularly bead mill types arelimited as to production capacity, i.e., the number of specimen vesselsthat can be oscillated at one time. For example BEAD BEATER bead millsmanufactured by BioSpec Products of Bartlesville, Okla., for a long timeonly could be used to oscillate one specimen at a time, althoughrecently a bead mill for use with up to eight specimen vessels at onetime has been introduced. These bead mills either single or pluralspecimen holding, operate to reciprocate the specimen holding vesselshorizontally with respect to a horizontal axis defined by a rapidlyrotating shaft that drives the oscillating mechanism. Where pluralspecimen vessels are oscillated together, they have been clustered closeabout the horizontal axis. A disadvantage of that arrangement is thatreproducibility of oscillating conditions to be the same in each vesselis difficult, if at all possible, to achieve. Where a separationprotocol is to be practiced, conditions occurring in each specimenshould be replicated identically in each.

Oscillating a cluster of specimen vessels along a horizontal or nearhorizontal axis and involving use of bead mills of the above descriptionpresents serious balance problems in the oscillation producing mechanismcreating destructive effects leading to abort mechanism service life,the effect of horizontal oscillation on the mechanism bearing unit, forexample, being most extreme.

Another shortcoming of known bead mills is lack of capacity to produceoscillations greater than about 2800 oscillations per minute (about 46Hz). As a result, these bead mills are not capable of efficientlydisrupting tissues, particularly tissues having a medium or hardstructure and cells of certain types, and hence resort must be had tochemical lysing.

In dealing with the quest for improving mechanical lysing of tissues forrelease of cellular components, particularly nucleic acids, it is seenthat an apparatus that allows simultaneous separation of plural samplesat very high oscillating rate while maintaining optimum balance in theapparatus is required, this being attributable in part to understandingthat to combine high oscillation rate with high average linearacceleration in the material is difficult, but necessary to practice thepresent invention.

The present apparatus more rapidly effects mechanical separation ofnucleic acids, and particularly DNA, from a source thereof and does sowithout adverse effect on the nucleic acid. The apparatus operates atspeeds as high as 166 hertz (Hz), i.e., about 10,000 oscillations perminute and is effective to impart average linear acceleration to asource material of up to about 450 times gravity (×g) or more therebyproducing relatively complete lysis and release of nucleic acids in atime period that can be as low as from about 3 seconds to about 5minutes where a specimen vessel of typically 100 microliters (ul) toabout 5 milliliters (ml) volume is used to contain the specimen(50-2,000 ul) and about 200 ul to 3 ml of liquid.

Briefly stated, there is provided by use of the apparatus describedherein a method for rapidly oscillating specimen containing vessels in anucleic acid recovery operation wherein controlled mechanical force isemployed to disrupt the cell walls and tissue structure of a tissue usedas a source of the nucleic acids. The disruption, or lysing, of thetissue by mechanical means involves accelerating the source material torelatively high g (acceleration imparted to a body by gravity acting ina vacuum being one g) levels in an oscillatory fashion in a short timeto expose it to an average linear acceleration that will producesufficient mechanical energy in the source material that produces thecell disruption or fracture to allow release of nucleic acids from theorganized structures of the cells of the tissue.

The apparatus includes a specimen vessel holder provided as a disc inwhich the vessels are received. The disc is operably connected withoscillatory motion producing means that in operation oscillates the discrapidly in an oscillatory movement up and down symmetrically on a fixedvertical axis. The disc is haltered so it cannot rotate about the fixedaxis. Locking means in the form of a locking plate locks the vessels onthe vessel holder and applies clamping force thereto to prevent relativemovement between the vessels and the holder so that generation of heatthat could be detrimental to the specimen material or the vesselsholding same is obviated.

The apparatus for rapidly reciprocally vibrating specimen-containingvessels accelerates a specimen material (tissue) in the vessels torelatively high g levels. In one embodiment, the apparatus includes adisc shaped vessel holder, the vessel holder having vessel receptivestructure arrayed thereon at a plurality of circularly spaced locationsproximal a disc edge periphery for receiving and holding up to acorresponding plurality of specimen vessels thereon. A verticallyoriented rotary shaft rotatable about a fixed axis has a mounting collarfixed thereon to rotate therewith. The mounting collar has an outersurface, this outer surface being symmetrical about an axis skewedlongitudinally of the fixed axis. The vessel holder is mounted on thecollar outer surface such that the vessel holder vessel receptivestructure is symmetrically arrayed with respect to the skewed axis andsuch that there is relative rotatability between the mounting surfaceand the vessel holder. When the mounting collar is rotated by rotaryshaft rotation and the vessel holder not held, it tends to rotate inunison with the mounting collar about the skewed axis but if the vesselholder is held against this tendency to rotate with the mounting collar,the vessel holder will be caused to oscillate vertically up and downsymmetrically of the fixed axis with any given point at the disc edgeperiphery undergoing one complete oscillation for each rotary shaftabout said fixed axis, as is means for haltering the vessel holder sothat it cannot rotate in unison with the mounting collar.

In another embodiment, the apparatus comprises a disc shaped vesselholder, along with a vertically oriented rotary shaft rotatable about afixed axis with the vessel holder being mounted on the rotary shaft suchthat there can be relative rotatability therebetween. Means are providedfor holding the vessel holder to constrain a rotation of the vesselholder if the rotary shaft is rotated. Oscillatory motion producingmeans is operably connected with the rotary shaft and the vessel holderand is operable such as to cause the vessel holder to oscillatevertically up and down symmetrically with respect to the fixed axis whenthe rotary shaft is rotated, any given point at an edge periphery of thedisc undergoing one complete oscillation for each rotary shaftrevolution. The disc shaped vessel has a circularly arrayed uniformlyspaced plurality of specimen vessel receptive openings therein locatedproximal the edge periphery of the vessel holder, with a center of eachopening being equidistant from the fixed axis whereby an oscillationproduced acceleration to which a material contained in a specimen vesselreceived in an opening is subjected, is substantially the same withrespect to that produced in a specimen vessel received in anotheropening.

The apparatus can subject the specimen material to oscillations at anoscillatory rate of between about 25 Hz to about 133 Hz and can producean average linear acceleration in the source material which is in arange of about 150×g to about 415×g for a period of between about 3seconds to about 5 minutes.

The apparatus uses a vessel or container useful for containing aspecimen material which is to be subjected to a specimen treatmentduring which treatment, the vessel and or specimen material can beexposed to heat that could be detrimental to specimen and/or vesselintegrity, this vessel being a sealable member having an inner specimencompartment for holding a specimen material, and an outer casingsurrounding the inner compartment in which a freezable or readily cooledfluid can be received so that when such fluid has been frozen or cooledto very low temperature and the contained specimen subjected to saidtreatment, the specimen in the inner compartment and the vesselstructure is temperature protected from heat produced incident thetreatment by preferential transfer of heat into the fluid. Means such asremovable caps for sealing an entry to each of the inner compartment andthe outer casing are provided.

Using the apparatus described herein, average linear accelerations canrange between from 150 g up to at least about 415 g or more. Further,oscillation rates of up to at least about 116 Hz to 133 Hz or more arepossible. A Hz is a unit of frequency, and 1 Hz is equal to one cycleper second. For example, 116 Hz corresponds to an oscillation rate of7000 and 133 Hz to a rate of 8000 cycles per minute.

In practicing a protocol it is convenient to use inexpensive, disposableplastic vessels or vials for holding the source material.

The apparatus is intended particularly for use in a laboratoryenvironment wherein it will be seated on a counter or table top readilyaccessible for use by the scientist or technician. For that reason itwill be housed in a casing having a cover, and since the apparatus isportable and of reasonable weight and size is readily movable from oneto another laboratory location without difficulty. The casing preferablywill be fitted with suction cups at the underside as these obviate anymovement action of the casing along a counter top during operation, andcaused by operation vibrations. To further diminish vibration effect,the apparatus is isolated from the casing by vibration absorbing means.

FIG. 1 depicts a casing C in which the apparatus 10 is housed. Thecasing C includes a cover 2 which is closed during the apparatusoperation, and it can be provided with safety interlock features suchthat the cover is locked and cannot be opened during operation and thatthe drive motor operating the apparatus cannot be activated unless thedoor is closed. Such features are considered essential to protectpersonnel and prevent injury from apparatus that operates at extremelyhigh speeds.

Within the casing, a fixed support drum 6 will mount the apparatusthrough the intermediate vibration absorbing anchor structure to bedescribed later. In this manner no serious or undesirable vibrationeffect will transmit from the operating apparatus to the casingstructure. The casing C also will mount controls such as switches, timerunit etc., these being shown generally at 4. Further, the casing caninclude a fan unit therein to circulate a stream of cooling air againstthe apparatus to carry off heat therefrom which is generated duringoperation and particularly in the bearing unit that will be describedlater.

With reference to FIG. 2, the apparatus 10 comprises a drive motor 12having a vertically oriented output or drive shaft 14 which is rotatableabout a fixed vertical axis, the motor being hung or suspended fromanchor structure shown generally at 18, the motor being capable ofrotating at speeds up to at least about 8000 R.P.M. The anchor structure18 includes a plate 21 and blocks 7 on which it is set, the blocks inturn being mounted on drum 6. Intervening the plate 21 and the blocks 7is a resilient material pad 20 which preferably is of rubber and onewhich exhibits stiffness in respect of a twisting thereof yet is readilyflexible and yielding in respect of vertical force applied thereto. Pad20 serves to damp vibrations transmitted through the plate 21 thatotherwise could enter the drum 6 and transmit to the casing C.

The upper part of the housing 8 of the motor 12 is connected to theplate 21 as by bolts 9 (only one shown) and in such manner the motor andthe remainder of the apparatus is suspended mounted thereby lesseningvibration generation in the apparatus and casing.

The single suspended mounting of the apparatus is particularly effectiveto the purpose of minimizing operation produced vibrations, this beingachieved with use of a single relatively thin disc shaped pad member 20and placement of the orientation of the pad member to be planarperpendicular to the fixed axis F. The pad member as noted above isselected as a rubber component exhibiting two stiffness. With respect totorque force circularly acting in direction perpendicular to axis F, thepad is extremely stiff which is desirable from the standpoint of dealingwith torque as a factor in vibration cause. On the other hand and withregard to force acting parallel to the axis F, the pad material is verysoft, i.e., has little stiffness so that the force is readily damped bythe flexibility of the pad in that force direction.

The apparatus includes oscillatory motion producing means showngenerally at 22, the oscillatory motion producing means being of a typesimilar to that used to produce a like motion in the earlier-mentionedBioSpec bead mills. Such means includes an eccentric mounting collar 11integral with a hub 13, this unit being screwed on to shaft 14 androtatable with shaft 14.

This oscillatory motion producing means also includes a bearing unitcomprised of an inner race 21 clamped between hub 13 and a nut 15threaded on shaft 14 so as to be fixed to rotate with the mountingcollar, an outer race 23 fixed to a central bore of a relativelywidened, relatively shallow vessel holder 24 made preferably in theshape of a disc located a distance above the anchor structure, and aplurality of ball bearings 19 captive between the races. A preferredform of bearing is a double row angular contour ball bearing.

The mounting collar 11 has an outer surface which is symmetrical aboutan axis K which is skewed longitudinally of the fixed shaft axis F. Thusit is seen that the vessel holder 24 is mounted on the mounting collarsuch that vessel holder vessel receptive structure (to be describedshortly) is symmetrically arrayed with respect to this skewed axis K.Further it is seen that relative rotatability exists between the vesselholder and the mounting collar.

With this arrangement, it is seen that if the vessel holder 24 not beheld during rotation of the mounting collar 11, the vessel holder wouldbe caused to have a certain rotation in unison with the mounting collarabout axis K, such rotation being at the inclined solid line showing ofthe vessel holder in FIG. 2. On the other hand, if the vessel holder 24is haltered or held during mounting collar 11 rotation, the vesselholder will be caused to oscillate vertically up and down andsymmetrically with respect of fixed axis F. This movement is illustratedin exemplary showing in dashed line vessel holder fragment positioningas at OS in FIG. 2.

It will be understood that this vertical oscillatory movement of thevessel holder occurs such that any given point at the periphery of thevessel holder will undergo one complete oscillation up and down eachtime shaft 14 and mounting collar 11 make one complete revolution.

Vessel holder 24 in a preferred form is a disc having a hub 25, a numberof arms 27 emanating from the hub and terminating in an annularperiphery ring 29. Annular periphery ring 29 it will noted is of muchlesser thickness than the thickness of radially inwardly parts of thevessel holder, this being desirable to reduce the mass of the holder.

Since considerable heat will be generated in the apparatus andparticularly in the bearing unit during operation, it is desirable thatthe disc mass function as a heat sink to carry off heat, the disc forthat reason being of a material which has good heat conductivitycharacteristic, aluminum being exemplary of such material.

The vessel holder 24 will have suitable structure thereon for receptionand holding of a plurality (e.g., at least 18) of specimen containingvessels, the depicted ones of such being scalable vials 26, the vialsbeing fitted with seal caps 28.

In simplest form, this holding structure can be constituted of a circleof uniformly spaced openings 32 carried in annular periphery ring 29 andpassing therethrough from one to an opposite face. In this manner a vialbody passes down through an opening 32 until its vial flange 47 engagesthe upper disc face adjacent the opening to hold the vial mounted on thedisc. Other forms of holding structure or devices could be used insteadof openings.

In connection with openings 32, a center of each is equidistant locatedfrom a center of the holder. In this manner, a specimen contained in avessel received in an opening will be subjected to the exact sameaverage linear acceleration values to which a specimen contained in avessel received in any other opening 32 is subjected ruing apparatusopening. It is to be noted that average linear acceleration imparted tothe specimen will be the same if only one vial is mounted on the vesselholder as that attendant mounting of a full complement of 18 vials onthe vessel holder.

This sameness of replication of achieved linear acceleration for eachseparation protocol of each specimen whether for one or for 18 specimensat the same time, and stemming from symmetrical positioning of vesselson the vessel holder is seen as a major improvement over priorseparating apparatus.

A halter means is used to prevent rotation of the disc 24 in unison withthe mounting collar 11 during apparatus operation. This halter means canbe, e.g., a tension type coil spring 3 connected to the disc at anyunderface part thereof and with the anchor structure 18, connection tothe anchor structure minimizing extraneous vibration transmission to thespring. The spring 36 will be connected to the underface of the disc 24at a radial location thereon which is closely proximal the shaft 14 andsuch that the spring disposes parallel to fixed axis F, this being doneto limit the degree of tensing produced in the spring thereby reducingfatigue effect and lengthening spring useful service life.

By haltering the disc 24, oscillatory motion producing means driveeffect thereon is as mentioned above to rapidly vertically oscillate thedisc, periphery of the disc ring describing an imaginary rolling wavecourse about the shaft 14, it being understood that there is no circulartravel of the shaft during oscillation thereof.

The result is that the vials 26 are rapidly oscillated in verticalreciprocal movements at a rate of as much as eight thousand oscillationsper minute (133 Hz). Due to that rapid oscillatory movement of the vial,average linear acceleration values of up to 415 g are produced in thevial contents and the small sized bead in the vial produce very highimpact magnitudes as they collide with the cells of nucleic acid sourcematerial therein and produce significant cell disruption to allownucleic acids to release from the cells.

Depending on the type of tissue source material involved, essentiallyfull release can be effected very quickly and in a time period rangingfrom about 10 to about 120 seconds and particularly in a range,depending on the material, of from about 10 to 30 seconds to about 30 to60 seconds.

Because of the nature of the oscillatory movement to which the vials 26are subjected, it is necessary to securely lock the vials on the discperiphery ring 29 so that during oscillation, no relative movementoccurs therebetween as such relative movement could create high frictionand consequent heat problems in the specimen and in the vessel.

To obviate such possibility, the locking of the vials is done with alocking plate 50 as shown in FIGS. 3 and 4. The locking plate 50 ismountable on top of the disc 24 and can be secured to the latter with anumber of locking members or hand manipulated knobs 52 threaded as at 55into passages in the disc, tightening of the knobs to friction holdingdegree locking the fixing plate against the disc.

As shown in respective clearing and covering dispositions in FIGS. 3 and4, the locking plate 50 has blind slots 51 therein so it is circularlymovable on the disc to accommodate loading/unloading of vials on thedisc on the one hand, and securely clamping the vials in place on thedisc on the other hand.

To securely hold the vials, the locking plate 50 has a circle of spacedradial fingers 54 in correspondence to the number of vial receptiveopenings in the disc. These fingers 54 when locking plate 50 is inlocking position, engage the top of the vial caps 28 and apply hold downforce to the vials. The urging is to forcefully hold the vial flange 47against the upper face of the disc periphery ring 29 adjacent theopenings 32 in the disc. This bars relative movement between the vialsand the disc during operation.

FIG. 5 shows another form of locking member 56 for clamping or lockingthe locking plate tightly against the vials and disc. It comprises aspring locking member unit which is depicted in unlocked position indashed lines. By rotating the locking member arm 58 to the solid lineposition, a camming hold down effect is instituted.

Other forms of haltering means can be used with the apparatus, thesebeing advantageous if spring fatigue is a problem with the earlierdescribed haltering means. FIGS. 6 and 7 depict a haltering means 70provided with permanent magnets. In such means 70, a bracket 72 carriedon the anchor frame mounts a permanent magnet 74, and a bracket 76carried on the underside of the disc 24 mounts a permanent magnet 78.These permanent magnets are arranged in a confronting disposition, andthe poles thereof arrange so that like poles face each other. Thiscreates a magnetic repelling force that acts against the disc 24 so thatif it tends to rotate in unison to any degree with the mounting collarduring apparatus operation, the magnet repelling force prevents suchdisc rotation. It is to be understood that at least one of the magnetmembers will be of greater vertical dimension than the other to takeinto account the relative vertical movement of the magnet mountingelements that occurs during oscillation.

FIGS. 8 and 9 show a still further form of haltering means comprised ofan upstanding post 80 carried on the anchor structure, and a passage 82formed through the disc 24. The post 80 extends through the disc passageso that rotative movement of the disc is effectively barred.

Where the haltering means is susceptible to failure, an occurrence morelikely where a resilient spring is used, it is important to provide abackup haltering means such as that 110 depicted in FIG. 1, such backupmeans being, e.g., the same as that depicted as a haltering means inFIG. 9.

FIG. 10 shows a vial 90 that includes an inner compartment 92 forholding specimen material, small sized beads, etc. A casing wall 94surrounds the outside of the inner compartment defining structureleaving a space 96 that can be filled with a heat transfer liquid suchas water. Caps 108, 109 are used to seal entry to the inner compartment92 and space 96. Prior to use, the vial can be placed in a freezer so asto chill the liquid which if water freezes to ice. When used, heatgenerated during oscillation of the vial can be absorbed by the fluid orice which acts as a heat sink drawing heat away from the vial structureand the contents.

In effecting nucleic acid separation, it generally is best effected byrapidly reciprocally oscillating the tissue source material in thepresence of bead-containing liquid medium at such a rate that producesan average linear acceleration in the source material which is in arange of about 150 g to about 415 g and at an oscillation rate betweenabout 50 Hz to about 133 Hz the period involved for effecting separationbeing one in a range of time between about 10 to 120 seconds. Manyprotocols can be practiced with effective result using an oscillatoryrate of about 100 Hz such as to produce average linear acceleration ofat least about 300 g for a period of between 10 to 60 seconds.

The apparatus is used in conjunction with novel containers forconducting the isolation processes of the invention. The containerscomprise a cover and a lower member for containing the extractant andother components, also referred to herein as the “holder”. The holdercan take a variety of forms both as to shape, size and material ofmanufacture, depending upon the intended use, which variables are notconsidered to be necessarily limiting to the invention, and which willbe apparent to one skilled in the art. For example, the cover mayalternately be considered a cap, lid, top, etc, and may attach byfriction, seal, threads, clamp, etc., and may be removable from thelower member.

The container used in the present methods can also vary, but in somecases it may be desirable for the container to have concave ends so asto conform to the shape of the sphere, as illustrated in FIG. 11A or11M. The advantages of conforming top and bottom ends are several,including increasing durability of the container during use byminimizing the stress to the ends during use, and increasing ruptureeffectiveness by removing dead “spaces” where larger tissue fragmentscan avoid impact by the bead.

In addition, the mechanism for securing the top to the container canvary, so long as the top is openable and yet can retain the contentsduring oscillation. Thus, the invention is not to be considered aslimited to any particular container as container design is not aprinciple focus of the present invention. All the container embodiments,e.g., A-D and L-M, illustrate a bead in a container, which mustnecessarily be configured with an openable top, although the details ofthe top(s) are not defined.

A further permutation is illustrated in FIG. 11M, showing an inner andouter container, in which the inner container holding the sphere alsohas small pores of preselected diameter as in a cage to allow materialout through the pores during the rupturing process to the extent of thepore diameter. This embodiment facilitates separation of the releasedsuspension, including nucleic acids from insoluble or indestructiblematerials in the tissue. In this embodiment, the outer containercollects the material which passed out through the pores, and “L”identifies a removable lid on the outer container.

In a particularly preferred embodiment, the container has substantiallycylindrical walls such that when utilized with a spherical bead theeffect is similar to a dounce homogenized, whereby the clearance betweenthe inner walls of the container and the surface of the sphere can beadjusted so as to define the thickness of the article to be disrupted.In preferred embodiments, the clearance is selected to be less than thediameter of a cell in the tissue to be disrupted, such that by use thecells are broken without disrupting subcellular organelles. In otherembodiments, the clearance is selected so as to disrupt both cell andnuclei without disrupting smaller subcellular organelles, such asmicrosomes and other vesicles that may contain nucleolytic enzymes.Thus, a clearance can be as small as the diameter of subcellularorganelles, or on the order of 10, 25 and 50 microns (u), on up to thediameter of small cells, such as 100 u (0.1 mm), and on up to thediameter of large cells, such as about 3 mm.

A preferred clearance useful in the present methods is in the order ofabout 25 microns (0.025 mm) to about 3 millimeters (mm), preferablyabout 0.8 to 1.5 mm, and more preferably about 1 mm. Of course, theclearance achieved is a function of both the container inner diameterand the sphere utilized. Preferred are 1 to 2 ml containers and sphereshaving about 5 to 10 mm diameters.

In another embodiment, it is appreciated that the container can containtwo or more spheres having different clearances for the purpose ofspecifically rupturing structures, tissues, cells and/or organelles in acoordinated manner. For example, whereas a very small clearance spheremay have difficulty initially with a crude sample, a large clearancesphere will rapidly break the sample into smaller diameter fragmentswhich the smaller clearance sphere may then productively homogenize.Thus the invention contemplates the uses of combinations of clearancesin two or more spheres.

For use in research and other laboratories where relatively smallamounts of DNA are required, the containers can be packaged in kitscontaining one or a plurality of containers together with containers forbuffers, reagents and other accoutrements appropriate to the practice ofthe present invention. The kits may further include a selection ofcontainers with particles of different sizes and/or densities toaccommodate the varying sizes of the cells or hardness of tissuesemployed as the DNA source material. Such containers are especiallyuseful with an apparatus which can hold a plurality of containers, evenup to 20 or more. Such machines and containers are especially usefulwhen it is desired to conduct a number of DNA isolations simultaneouslyor sequentially.

2. Methods for Isolation of Nucleic Acid from Tissue

The present invention describes a method for disruption of tissues tofacilitate release and ultimately recovery of selected cellularcomponents, particularly nucleic acids, and more particularly DNA, in apurification procedure for those components.

The invention involves subjecting the tissue in a liquid medium tomechanical energy of a particular type as specified herein so as todisrupt tissue and cell structure sufficiently to release nucleic acids,and particularly DNA, into the liquid phase for subsequent recovery andpurification.

As described herein, the choice of mechanical energy and disruptionconditions depends upon the type of tissue to be disrupted, and theprocess may involve the use of one or more particles to assist theapplication of mechanical energy.

In addition, the release by mechanical energy is conducted by combininga tissue containing the DNA with a liquid medium in a closed containersuitable for applying the mechanical energy.

The nucleic acid source material can be any source believed to containnucleic acids, including bacteria, fungi or yeast cells, viruses, plantor animal tissue, foodstuffs, gels, process by-products, soil or watersamples, industrial solutions, and the like materials having nucleicacids. The nucleic acid source material, cell or tissue can range instructural complexity, subcellular organelle content, and level oftissue organization, which differences contribute to the structuralintegrity, i.e., “hardness” or “softness” of the material from amechanical disruption perspective, as described further herein.

The nucleic acid source material is typically provided as a paste orpellet if provided as a bacteria, fungi, yeast or any cultured cells,and as pieces of tissue in small fragments if derived from plants oranimals. For example, single-cell suspensions of bacterial or yeast aretypically provided by centrifugation or filtration to yield a pellet ora paste, which is conveniently transferred to a container as describedherein suitable for applying the oscillatory mechanical energy.

In the case of plants or animals, the particular portion of thematerial, e.g., muscle, brain, kidney, etc., or leaf, seed, root, stem,etc., is collected, and may be fragmented to a convenient size of about0.1 mm to 2 cm by a variety of methods including surgical sectioning,smashing to randomly break the tissue, fragmentation by freezing thetissue and then rapidly impacting the frozen tissue to shatter it intopieces, and the like fragmentation methods.

Freezing and shattering is particularly preferred because of thebenefits of maintaining the provided biological tissue cold. Freezing istypically effected by immersion of the provided tissue into liquidnitrogen, or contacting the tissue with dry ice, until frozen. Theshattering is typically effected by placing the frozen tissue into aplastic bag or foil container, and impacting the frozen tissue with ahammer with sufficient force to shatter the tissue into pieces.

The liquid medium is formulated to assist the disruption process, butmay also contain materials to assist the recovery process. The liquidmedium is typically a buffered cell resuspension solution. Exemplaryliquid media are described further herein.

The total volume of the container used for applying the mechanicalenergy to the DNA source material should be sufficient so that when itis closed, it will hold the liquid medium, the DNA source material andthe other components under conditions so that the entire mixture can beconveniently and efficiently shaken. A general rule for this purpose isthat the total volume of the closed tube is about two-thirds (⅔)tissue/buffer and about ⅓ air space. If the container further containsparticles to aid the mechanical lysis, the total volume of the closedtube is about ⅓ particles, ⅓ tissue/buffer, and about ⅓ air space.

In particular, the amount of particles can be an amount that occupies avolume approximately equal to about 1 to 100% of the liquid mediumvolume, although volumes of about 5 to 80%, and particularly about 10 to50%, are more preferred.

DNA release from the cell or tissue structure of the source material iseffected by the application of the specified mechanical energy for apredetermined time period. The time period of applied mechanical energyrequired depends principally upon the type of source material, the“hardness” of the tissue, and the size of the source from which the DNAis being extracted since these parameters for the various DNA sourcessuch as bacteria, yeasts and plant or animal varies appreciably.

Time is not a particularly critical factor so long as a sufficientamount of time is used such that most of the DNA is released from thesource, but not excessive time used so as to prevent excessive shearingof the DNA to be isolated. The particular time period used can bedetermined empirically by preparing samples of the material under one ormore of the preferred conditions defined herein depending on the“hardness” of the source material. Exemplary times are described hereinand in the Examples.

Following the release of the DNA into the liquid phase, any of a varietyof DNA recovery methods may be used, including, but not limited toadsorption to a solid support, enzymatic treatment combined withselective precipitation, organic extraction, and the like methodsdescribed further herein.

Since rupture of the cell walls can release all of the cellularsubstituents, this invention can be used with or without chaotropicagents and extraction solvents such as those described herein to isolateother cellular components using known isolation procedures. For example,proteins may be isolated from a disrupted mixture containing anextraction solvent that comprises a neutral buffer and a cocktail ofprotease inhibitors.

As another example, the processes and containers of the invention may beused to efficiently and rapidly shred tissue such as skin, intestine,gastric, liver etc. into the component parts for the isolation ofcertain components. Individual cellular components, e.g., enzymes, maythen be isolated using standard chromatographic techniques. Similarly,structural components e.g., connective tissue, membranes, cell wallcomponents, etc. may be separated by differential centrifugationtechniques.

3. Particles for Lysing Tissue

The invention deals with a method and apparatus specially suited fornucleic acid separation from its source material by subjecting thatmaterial to controlled mechanical energy as specified herein.

In one embodiment, the mechanical energy is applied in combination withparticles of varying size, shape and density in the liquid mediumcontaining the source material. It is believed that the presence of theparticles increases the mechanical energy applied to the tissues, andprovides a means for impacting, striking, breaking and/or rupturing thetissue so as to facilitate release of nucleic acids from the tissue andthe DNA isolation process.

Any convenient number or weight of such particles may be employed,although the particular number and weight of particle somewhat dependsupon the size and shape of the particle, and also on the particulartissue being treated, with the end objective of selecting a mechanicallysing force sufficient to release nucleic acid without compromising thequality of the recovered product.

The shape of particle may vary, including spherical, elliptical,rectangular, irregular, and the like shapes. Therefore, except forpreferred embodiments, the terms “particle” and “bead” are usedinterchangeably to connote that various shapes may be utilized in thepresent invention. Exemplary shapes are shown in FIGS. 11A-11O.

The size of the particle also may vary depending on tissue type andscale of process, although particularly preferred are particles of fromabout 0.1 millimeter (mm) to about 2.0 centimeter (cm), and morepreferably about 4 mm to 8 mm. In particular embodiments, it may bedesirable to select the size of the bead relative to the container inwhich mechanical energy is directed, such that the clearance between thebead and the container internal wall defines the maximum diameter oftissue organelles that remain intact in the procedure, analogous to aDounce homogenizer. Thus, FIGS. 11A-11C represent three different sizedspheres (A-C) in which sphere A would homogenize to smaller sizes thansphere C, and sphere B would be intermediate, due to the respectivelygreater clearance in the container between sphere and container wallobserved when using spheres A-C, respectively. It is seen that the beadsize is dependent upon the scale of the procedure and the correspondingsize of the container in which tissue sample, liquid medium and particleare to be oscillated.

Exemplary particle shapes besides spheres are illustrated in FIGS.11I-11K, where 11I illustrates “odd” shapes with smooth edges and sides,11J illustrates irregular shapes with non-smooth edges, and 11Killustrates the irregularly shaped particles of 11J in a smaller sizeand used as a cluster.

Beads used in the protocol can vary in density, which provides certainadvantages. Beads that are relatively more dense provide the advantageof delivering relatively higher oscillation average linear accelerationforces to the specimen tissue which is advantageous where the “hardness”of the tissue to be ruptured is to be considered. Examples of the use ofvarying densities, e.g., plastic, glass, dense ceramic and steel, aredescribed herein and demonstrate usefulness depending on the hardness ofthe tissue structure.

Preferred plastic beads are constructed of teflon, polypropylene or PVC.Preferred ceramic beads are zirconium silica oxide ceramic or siliconnitride ceramic. Metal beads should be corrosion resistant, andstainless steel is preferred.

Beads may also vary in porosity, as illustrated in FIGS. 11E-11H, wheresphere E is solid, sphere F has fine pores, sphere G has medium pores,and sphere H has large pores.

B. Tissue Lysis Conditions for Varying Tissues

The most important feature of the preselected mechanical releaseconditions is that the conditions are capable of generating enoughmechanical energy by reciprocal motion to break the tissue structure andcell walls and release the nucleic acids.

Whereas for soft tissues, efficient release may be accomplished solelyby the mechanical forces upon the tissue in a liquid medium, other morestructured tissues are ruptured by subjecting the tissue to rapidlyoscillating particles or other inert particles in the liquid medium inthe presence of the tissue. Such particles are commercially available ina variety of sizes from several sources as described further herein. Thetissue, medium and particles are oscillated under pre-selectedconditions depending on the tissue type to provide sufficient mechanicalenergy to disrupt the tissue and cell walls.

It is important to emphasize that for the isolation of DNA, the use ofexcessive mechanical energy is undesirable because it will shear the DNAto low molecular weight lengths that are not desirable.

1. Tissue Types

The process of the invention is applicable not only to biological tissuesuch as animal or plant tissues, but also to microorganisms such asbacteria, viruses, yeast, fungi, mold and the like materials as sourcesof DNA. Such sources, especially bacteria, yeast and plants are muchmore convenient than animal tissue as a source of DNA because they canbe more uniform, are readily available in any desired quantities and canbe easier to work with than animal tissue based on uniformity andquantity.

More important, however, is the consideration of the “type” of DNAsource material used in the present methods. Because the structuralintegrity of the material, either at the level of subcellularorganelles, cell walls or tissue structure, can vary depending on thetype of material, the “hardness” of the material will also vary,affecting the choice of conditions under which the mechanical energy isapplied to release high molecular weight DNA from the tissue/cell ornon-cellular material.

For convenience, the “hardness” of a material or tissue can be brokendown into four groups, termed “hard”, “medium hard”, “medium soft” and“soft” to connote a gradation between the most structurally intactmaterials/tissues that are relatively the most resistant to mechanicallysis, to the least structurally intact tissues that are relatively theleast resistant to mechanical lysis.

A “soft” tissue is typically spleen, brain, liver, lymph, bone marrow,leukocytes, nucleated red blood cells, tissue cultured cells, softfoodstuff, gel, water sample, and the like soft tissues.

A “medium soft” tissue is typically kidney, heart, muscle, bloodvessels, tumor or tissue biopsies, immature plant tissue such as fruit,flowers, sprouts, young leaves, nematodes such as Caenorhabditiselegans, gram negative bacteria such as Escherichia coli, gram positivebacteria such as Staphylococcus aureus, Salmonella typhimurium, orMycobacterium tuberculosis, medium soft foodstuff, and like medium softtissues.

A “medium hard” tissue is typically skin, cartilage, soft bone, tailsnips (mouse tail), mature plant tissue such as mature leaves, tubers,legumes, chitinous tissues including whole insects such as mosquitos orfruit fly, slime mold such as Dictyostelium discoideum, yeast such asSaccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris,fungi such as Cryptococcus sp., algae, medium hard foodstuffs, and likemedium hard tissues.

A “hard” tissue is typically plant seeds or bark, plant and tree trunks,stems, rice, soybean, oats, corn leaf, kernels, grain such as Triticumaestivum roots and other woody materials, bones, hard foodstuffs, soilor fossil samples, and like hard tissues.

The assignment of a tissue to a particular “hardness” is not to beconstrued as absolute as some tissues can vary in hardness depending onthe condition of the source material. Therefore, in circumstances wheremechanical release is not efficient, or alternatively is overlydisruptive to the detriment of the released DNA, the “hardness”conditions should be varied empirically according to the variousprotocols described herein.

2. Lysis Conditions

DNA source material can be subjected to the mechanical energy accordingto the present invention under a variety of conditions designed todisrupt the tissue or cell structure and release the DNA into the liquidmedium. The conditions will vary depending upon the hardness of the DNAsource material to be treated, although certain aspects of thedisruption process can be readily varied for efficient release as willbe apparent to a skilled practitioner.

For example, the liquid medium may contain salts, buffers, stabilizers,detergents, and the like reagents.

Any of a wide variety of well known buffers which will permit control ofthe pH within the preferred ranges of about 5-9, preferably about6.5-7.5, and more preferably about 7.0, may be employed. Buffers basedon tris(hydroxymethyl)aminomethane (i.e., Tris), sodium acetate orsodium citrate are presently preferred because they are readilyavailable and provide excellent results. Other buffers known to theskilled artisan may be used.

For research purposes, which normally require only small amounts of DNA,the amount of DNA source material, liquid medium and container may bevery small. Typically, the total container volume is from about 1.0-3.0ml. Larger containers may be employed to obtain greater quantities ofDNA.

A preferred cell resuspension buffer for the disruption process containsbuffer from about 5-500 millimolar (mM), and preferably is Tris-HCl. Thebuffer preferably contains EDTA in an amount of from 0.5-500 mM. Aparticularly preferred buffer is comprised of 50 mM Tris-HCl, pH 7.0, 20mM EDTA.

A detergent may be included in the cell resuspension buffer. Typically,the detergent is included in the disruption procedures for morestructured tissues, such as medium soft, medium hard and hard tissue tominimize DNA damage during the release procedure. It has been determinedthat detergent in the liquid medium during application of the mechanicalenergy prevents excessive shearing of DNA such that the isolated DNA isof high quality for subsequent use in recombinant DNA manipulations suchas the polymerase chain reaction (PCR) and the like methods. Detergentsare typically not employed to facilitate disruption of soft tissues,particularly where disruption is conducted in the absence of particles,as described further herein.

a. Disruption of Soft Tissue

Typical disruption conditions for soft tissues are relatively moregentle, and include the use of the above described preferredTris-HCl/EDTA buffer in the liquid medium in a ratio of about 1:1 (v/v)material to liquid medium, and the application of about 25-75 hertz (Hz)oscillatory mechanical energy, more preferably about 50 Hz, to produce agravitation of about 100-200 times gravity (×g), more preferably about150×g, for a time period of about 5 to 60 seconds, preferably about 10to 30 seconds, and more preferably about 20 seconds.

b. Disruption of Medium Soft Tissue

Typical disruption conditions for medium soft tissues are relativelymore rigorous than for soft tissues, and include the use of the abovedescribed preferred Tris-HCl/EDTA buffer in the liquid medium in a ratioof about 1:1 (v/v) material to liquid medium, and the application ofabout 75-125 hertz (Hz) oscillatory mechanical energy, more preferablyabout 100 Hz, to produce a gravitation of about 200-400 times gravity(×g), more preferably about 300×g, for a time period of about 5 to 60seconds, preferably about 20 to 40 seconds, and more preferably about 30seconds.

For medium soft tissues, the application of mechanical energy ispreferably conducted in the presence of one or more particles to assistthe application of mechanical energy. Preferably, the particles usedoccupy a volume approximately equal to the volume of liquid medium suchthat the particle:liquid:material ratio is about 1:1:1 (v/v/v), althoughthe ratio of particle to liquid can be from about 0.2:1 to about 2:1.

In preferred embodiments, the particle used is a sphericle bead asdescribed herein, typically about 2-10 mm in diameter, although theprecise diameter depends upon the container such that there is to beclearance of at least 0.5 mm, preferably about 1 mm, between the wallsof the container and the sphere. In a preferred embodiment, thecontainer has an inner diameter of 8 mm and the sphere is about 7 mm.

For disrupting medium soft tissue, it is also preferred that theparticle be of relatively low mass so that the impacts delivered duringapplication of the oscillatory mechanical energy are of relatively lowmomentum. Typical mass would be that provided by a non-brittle plasticsphere such as polypropylene and the like plastics.

Furthermore, for disruption of medium soft tissue to release highmolecular DNA, it is preferred to include a detergent in the liquidmedium at a concentration of about 0.1 to 10% weight per weight ofliquid medium (w/w), preferably about 0.1 to 5%, more preferably about0.5 to 3%, and more preferably about 1-2%. Typical detergents useful inthe method are described herein, although particularly preferred is theuse of 1 to 2% SDS in the liquid medium.

c. Disruption of Medium Hard Tissue

Typical disruption conditions for medium hard tissues are relativelymore rigorous than for medium soft tissues, and include the use of theabove described preferred Tris-HCl/EDTA buffer in the liquid medium in aratio of about 1:1 (v/v) material to liquid medium, and the applicationof about 75-125 hertz (Hz) oscillatory mechanical energy, morepreferably about 100 Hz, to produce a gravitation of about 200-400 timesgravity (×g), more preferably about 300×g, for a time period of about 5to 60 seconds, preferably about 20 to 40 seconds, and more preferablyabout 30 seconds.

For medium hard tissues, the application of mechanical energy ispreferably conducted in the presence of one or more particles to assistthe application of mechanical energy as was described above for mediumsoft tissues, with the following exceptions.

For disrupting medium hard tissue, it is preferred that the particle beof a medium mass so that the impacts delivered during application of theoscillatory mechanical energy are of relatively average momentum.Typical mass would be that provided by a non-brittle ceramic sphere suchas Zirblast (Specialty Ball Co., Rochy Hill, Conn.) and the likeceramics.

Furthermore, for the disruption of medium hard tissue, it is preferredto include a detergent in the liquid medium as described above formedium soft tissues.

d. Disruption of Hard Tissue

Typical disruption conditions for hard tissues are relatively morerigorous than for medium hard tissues, and include the use of the abovedescribed preferred Tris-HCl/EDTA buffer in the liquid medium in a ratioof about 1:1 (v/v) material to liquid medium, and the application ofabout 75-125 hertz (Hz) oscillatory mechanical energy, more preferablyabout 100 Hz, to produce a gravitation of about 200-400 times gravity(×g), more preferably about 300×g, for a time period of about 5 to 120seconds, preferably about 30 to 60 seconds, more preferably about 40seconds.

For hard tissues, the application of mechanical energy is preferablyconducted in the presence of one or more particles to assist theapplication of mechanical energy as was described above for medium softand medium hard tissues, with the following exceptions.

For disrupting hard tissue, it is preferred that the particle be of ahigh mass so that the impacts delivered during application of theoscillatory mechanical energy are of relatively high momentum. Typicalmass would be that provided by a metal sphere such as steel and the likerelatively hard metals.

Furthermore, for the disruption of hard tissue, it is preferred toinclude a detergent in the liquid medium as described above for mediumsoft tissues.

3. Detergents

In preferred embodiments, particularly for the disruption of mediumsoft, medium hard and hard tissues, the liquid medium used for theapplication of oscillatory mechanical energy includes a detergent in therange of about 0.1 to 10% (w/v), preferably about 0.1% to 5%, morepreferably about 0.5% to 3%, and still more preferably about 1 to 2%.

The selected detergent may be any of a variety of conventionalsurfactants including anionic, cationic, non-ionic and amphotericsurfactants.

Typically useful anionic detergents include, for example, sodium dodecylsulfate (SDS), sodium-n-decyl sulfate and triethanolamine dodecylbenzene sulfonate.

Cationic detergents useful in the practice of the invention include, byway of example, cetyl trimethyl ammonium bromide and other N-alkylquaternary ammonium halides, we well as polyethoxylated quaternaryammonium chloride.

Amongst the nonionic detergents, there are tallow fatty alcoholethoxylates, ethoxylated tridecyl alcohol, ethoxylated tridecanol, nonylphenol ethoxylate and octylphenoxy polyethoxy ethanol.

Amphoteric detergents include, for example cocoamidopropyl betaine,disodium tallowimino diprioionate and cocoamido betaine.

A particularly preferred surfactant is SDS.

All of these detergents, and many other equivalent surfactant compoundsare readily available from commercial sources.

It is emphasized that the use of detergent is particularly preferred forthe isolation of high molecular weight DNA from medium soft, medium hardand hard tissues. Based on the results shown in the Examples herein, itis seen that the shearing of DNA is excessive in the absence ofdetergent for isolation of DNA from the harder tissue, whereas lysis ofsoft tissue in the presence of detergent produces little or no lysis.

C. DNA Recovery Methods

Following release of the DNA into the liquid medium by the applicationof oscillatory mechanical energy, the released DNA can be recovered byany of a variety of well known DNA isolation methods. In this regard,the invention is not to be construed as limiting, although severalpreferred recovery methods are described.

Exemplary DNA recovery methods include (1) adsorption onto a solidmatrix, such as silica, latex or polystyrene, followed by selectivewashing and elution of the washed DNA, (Sambrook et al., “MolecularCloning: A Laboratory Manual” 2nd Ed., Cold Springs Harbor Press, 1989;and Reddy et al., “Current Protocols in Molecular Biology”, 4.4.1-4.4.7,Ausebel, F. M., et al., Eds., Wiley, New York, 1991), (2) enzymatictreatment to digest protein and RNA, followed by salting out to removeprotein and detergent (GNOME DNA ISOLATION KIT, Cat. No. 2010-200,BIO101, Inc., Vista, Calif.) and (3) extraction with organic solvents(Sambrook et al., supra, and Reddy et al., supra).

The following examples are given by way of illustration only and shouldnot be considered limitations of this invention, many apparentvariations of which are possible without departing from the spirit orscope thereof.

EXAMPLES

1. Reagents for Use in the Methods

The following reagents were prepared and used in practicing the methodsof the invention.

A. Cell Resuspension Solution: 50 mM Tris-HCl, pH 7, 20 mM EDTA.

B. RNAse Solution: 50 mM Tris-HCl, pH 7, 5 mM EDTA, 5 mg/ml RNAse A.

C. Cell Lysis/Denaturing Solution: 1% SDS in Cell Resuspension Solution.

D. Protease Solution: 5 mg/ml Proteinase K, 5 mg/ml Pronase in CellResuspension Solution with 1% SDS.

E. Saltout Solution: 5 M NaCl.

F. Acetate Solution; 5 M potassium acetate.

G. Binding Matrix: 30% (V/V) silica matrix granules in 6 M guanidinethiocyanate.

H. Wash Solution: 10 mM Tris-HCl, pH 7, 1 mM EDTA, 100 mM NaCl, 50%ethanol.

I. 10% SDS in water.

2. Release of DNA from Intact Mouse Liver Tissue

Mouse liver was obtained fresh, and quick-frozen on dry ice. Thereafter,the frozen liver was smashed with a hammer into small tissue fragments,typically of about from 3 cubic millimeters (mm³) to about 0.1 mm³.Alternatively, fresh liver was sectioned to about 3-0.1 mm³ fragments,and used directly.

One hundred milligrams (mg) of frozen liver tissue fragments or fresh,unfrozen tissue were weighed out directly into a 2.0 ml microcentrifugetube (PGC Scientific, Gaithersburg, Md., Cat. #16-8115-34), 1.0 ml ofCell Resuspension Solution was added, and the resulting mixture wassubjected to oscillatory mechanical energy in the oscillation apparatusdescribed herein in the amount of 75 Hz producing about 200×g for 20seconds to form a solution of disrupted cell components, includingreleased DNA.

Liver tissue is considered a “soft” tissues and when treated in thismanner can be readily disrupted by the above conditions to release theirDNA for further isolation. DNA can also be prepared in this manner frombrain, lymph, marrow, tissue cultured cells, non-tissue sources such asgels, soft foodstuffs, soil or water samples, and the like softmaterials as described herein.

The resulting solution containing released DNA is then isolated in pureform from the other released cellular components by conventional DNAisolation methods. Exemplary are the two methods described herein usingadsorption to silica particles described in Example 6, or using anenzymatic method as described in Example 7.

3. Release of DNA from Intact Mouse Kidney Tissue

Mouse kidney was obtained fresh, and quick-frozen on dry ice.Thereafter, the frozen kidney was smashed with a hammer into smalltissue fragments, typically of about from 3 cubic millimeters (mm³) toabout 0.1 mm³. Alternatively, fresh kidney was sectioned to about 3-0.1mm³ fragments, and used directly.

One hundred milligrams (mg) of frozen kidney tissue fragments or fresh,unfrozen tissue were weighed out directly into a 2.0 ml screw-cappedmicrocentrifuge tube having substantially parallel walls of diameter 8mm available from PGC Industries. A polypropylene sphere of 7 mmdiameter available from Engineering Laboratories, Inc., N.Y., N.Y., and1.0 ml of Cell Resuspension Solution containing 1% (w/v) sodium dodecylsulfate (SDS) were added to the tissue fragments, and the resultingmixture was subjected to oscillatory mechanical energy in theoscillation apparatus described herein in the amount of about 100 Hzproducing about 300×g for 30 seconds to form a solution of disruptedcell components, including released DNA.

Kidney tissue is considered a “medium soft” tissue and when treated inthis manner can be readily disrupted by the above conditions to releasetheir DNA for further isolation. DNA can also be prepared in this mannerfrom heart, muscle, immature plant tissue such as fruit, sprouts, youngleaves, gram negative or gram positive bacteria, and like medium softmaterials as described herein.

The resulting solution containing released DNA is then isolated in pureform from the other released cellular components by conventional DNAisolation methods. Exemplary are the two methods described herein usingadsorption to silica particles described in Example 6, or using anenzymatic method as described in Example 7.

4. Release of DNA from Intact Mouse Skin Tissue

Mouse skin was obtained fresh, and quick-frozen on dry ice. Thereafter,the frozen skin was smashed with a hammer into small tissue fragments,typically of about from 3 cubic millimeters (mm³) to about 0.1 mm³.

One hundred milligrams (mg) of frozen skin tissue fragments or fresh,unfrozen tissue were weighed out directly into a 2.0 ml screw-cappedmicrocentrifuge tube having substantially parallel walls of diameter 8mm available from PGC Industries. A ceramic sphere of 7 mm diameteravailable from Specialty Ball Co., Rocky Hill, Conn., and 1.0 ml of CellResuspension Solution containing 1% (w/v) sodium dodecyl sulfate (SDS)were added to the tissue fragments, and the resulting mixture wassubjected to oscillatory mechanical energy in the oscillation apparatusdescribed herein in the amount of about 100 Hz producing about 300×g for30 seconds to form a solution of disrupted cell components, includingreleased DNA.

Skin tissue is considered a “medium hard” tissue and when treated inthis manner can be readily disrupted by the above conditions to releasetheir DNA for further isolation. DNA can also be prepared in this mannerfrom cartilage, soft bone, yeast cells, mature plant tissue such asmature leaves, tubers, legumes, chitinous tissues including wholeinsects, and like medium hard materials as described herein.

The resulting solution containing released DNA is then isolated in pureform from the other released cellular components by conventional DNAisolation methods. Exemplary are the two methods described herein usingadsorption to silica particles described in Example 6, or using anenzymatic method as described in Example 7.

5. Release of DNA from Intact Plant Seeds

Seeds were obtained fresh from wheat and quick-frozen on dry ice.Thereafter, the frozen seeds were smashed with a hammer into smalltissue fragments, typically of about from 3 cubic millimeters (mm³) toabout 0.1 mm³.

One hundred milligrams (mg) of fragmented seeds were weighed outdirectly into a 2.0 ml screw-capped microcentrifuge tube havingsubstantially parallel walls of diameter 8 mm available from PGCIndustries. A steel sphere of 6 mm diameter available from Abbott BallCo., Elmwood, Conn., and 1.0 ml of Cell Resuspension Solution containing1% (w/v) sodium dodecyl sulfate (SDS) were added to the tissuefragments, and the resulting mixture was subjected to oscillatorymechanical energy in the oscillation apparatus described herein in theamount of about 100 Hz producing about 300×g for 40 seconds to form asolution of disrupted cell components, including released DNA.

Plant seeds are considered a “hard” tissue and when treated in thismanner can be readily disrupted by the above conditions to release theirDNA for further isolation. DNA can also be prepared in this manner fromplant bark, plant and tree trunks, roots and other woody materials,bones, rice, and like hard materials as described herein.

The resulting solution containing released DNA is then isolated in pureform from the other released cellular components by conventional DNAisolation methods. Exemplary are the two methods described herein usingadsorption to silica particles described in Example 6, or using anenzymatic method as described in Example 7.

6. Recovery of DNA Using Silica Adsorption

The silica binding matrix was silica obtained from BIO101, Inc. (Vista,Calif.) in the form of “Glassmilk®”. The matrix comprises crushed silicaparticles having a range of sedimentation rate through still water atunit gravity of from 0.001 to 0.01 centimeters per minute (cm/min), anaverage size of from 0.5 to 8 microns, and a total size range of about0.2 to 20 microns. The binding matrix was provided as a 30% (v/v)suspension in 6 M guanidine thiocyanate.

For DNA suspensions that do not contain SDS, such as the suspensionprepared in Example 2, above, SDS was added from stock solution toproduce a suspension with 1% SDS. The DNA suspensions containing SDSprepared as in Examples 3-5, above, were processed as follows withoutfurther treatment.

About 600 microliters of the DNA suspensions containing SDS weresubjected to microcentrifugation at 15,000 rpm for 2 minutes to settleinsoluble and precipitated materials. Thereafter, 350 ul of 5 Mpotassium acetate solution was added to precipitate SDS and protein, thesuspension was mixed with the acetate by inverting the tube, and themixture was microcentrifuged as before for 5 minutes to produce adetergent-free supernatant.

500 ul of the resulting detergent-free supernatant, by either methods,was then transferred to a 800 ul spin filter centrifuge tube (SpinModule™ centrifuge tube, BIO101, Inc., Vista, Calif.) and 300 ul ofsilica binding matrix was added. Thereafter, the spin tube wasmicrocentrifuged as before for 2 minutes, and the flow-through in thedecant trap of the spin tube was emptied. 700 ul of Wash Solution wasadded to the spin tube, and the tube was microcentrifuged as before for2 minutes. The flow-through in the decant trap was emptied, and the spintube was again microcentrifuged for 2 minutes to remove all excessliquid from the binding matrix. The spin filter was then transferred toa clean trap tube, 100 ul of water was added to the filter, the bindingmatrix was suspended in the water by flicking the tube, and the spintube was then microcentrifuged as before for 2 minutes. The flow-throughcontained the eluted, isolated DNA in pure water.

7. Recovery of DNA Using Enzymatic Methods

DNA released into solution by the above described mechanical methods canalso be recovered in pure form (isolated) by using selective enzymaticdegradation of RNA and protein followed by salting-out the DNA.

To that end, to 1.0 ml of oscillated cell suspension from Examples 2-5is added 50 ul of RNAse Solution, and the mixture is thoroughly mixed.Thereafter, 150 ul of 10% (w/v) SDS is added and thoroughly mixed ifthere was no SDS previously added, and the mixture is incubated at 55-65degrees Centigrade (C) for 10 minutes. Thereafter, 35 ul of ProteaseSolution is added and thoroughly mixed, and incubated at 55 C. for 10minutes, inverting occasionally. Thereafter, 450 ul of 5 M NaCl is addedand thoroughly mixed to precipitate the SDS and proteins, and themixture is microcentrifuged as before for 10 minutes at 4 C. Theresulting clear supernatant is then removed with a large bore pipettetip and mixed with 1 ml water in a 15 ml tube, 4 ml of 100% ethanol isadded, and the tube is slowly inverted end to end to precipitate theDNA. The resulting DNA is then spooled out of solution, dried, andredissolved as needed to yield isolated, pure DNA.

8. Effect of Detergent and Particles on DNA Isolation Method Using SoftTissue

The DNA isolation method was carried out essentially as described inExample 2, except detergent and particles were varied to demonstrate theoptimal mechanical energy conditions.

To that end, 100 mg of frozen rat liver was placed in each of four 2 mlmicrocentrifuge tubes as described earlier, and designated tubes A-D. A5 mm diameter×3 mm width polypropylene disc was added to tubes B and D.One ml of Cell Resuspension Solution was added to each tube, and 100 ulof 10% SDS was added to tubes C and D, to produce 1% SDS finalconcentration. The clearance between the polypropylene disc and thecentrifuge tube inner wall was about 3 mm when measured at the widestangle for the disc in the microcentrifuge tube. The four tubes weresubjected to the same oscillatory mechanical energy in the apparatus asdescribed herein delivering 75 Hz and about 200×g for 20 seconds to forma solution of disrupted liver tissue, with the degree of disruptionvarying among the tubes.

FIG. 12 shows a picture of the four tubes containing the disrupted liversolutions (A-D), illuminated by back lighting to illustrate theturbidity. Tubes A and B showed considerably more turbidity, andtherefore more tissue and cell disruption than tubes C and D, indicatingthat the detergent almost completely inhibited tissue disruption, evenin the presence of a particle. Without detergent (tubes A and B), thedegree of lysis appears to be dramatically more extensive than withdetergent (tubes C and D). Furthermore, the turbidity is more extensivewhen no particle disc was used (tube A) than when a polypropylene discwas used (tube B).

Following mechanical energy disruption, the samples were subjected tocentrifugation and DNA isolation according to the enzymatic methoddescribed in Example 7. The isolated DNA was then analyzed for yieldsand quality by agarose gel electrophoresis. Equal aliquots ofDNA-containing samples produced from tubes A-D were electrophoresed andthen stained with ethidium bromide. The results of the electrophoresedDNA are shown in FIG. 13. Both yield and quality of the DNA samplesisolated in the absence of detergent are dramatically superior in termsof both amounts and higher molecular weight (samples A and B) whencompared to DNA isolated in the presence of detergent (samples C and D).Furthermore, the yield and molecular weight of the isolated DNA issuperior for DNA isolated in the absence of both detergent and aparticle (sample A) compared to isolation without detergent butincluding a particle (sample B). In particular, the yield of DNA forsamples C and D is estimated to be less than about 10% (by weight) ofthe amount isolated for sample A.

The results indicate that DNA isolated by the controlled mechanicalenergy method from soft tissue produces the highest yield and highmolecular weight quality when energy is applied in the absence of bothdetergent and particles.

9. Variations in Detergent for Isolation of DNA from Medium Soft PlantTissue

The effect of varying detergent concentrations during mechanical energydisruption of plant tissue for DNA isolation was analyzed. To that end,100 mg of freshly picked young grass leaf was admixed in each in sevenof 2 ml microcentrifuge tubes with 1 ml Cell Resuspension Solution andone 7 mm ceramic bead. Sufficient 10% SDS stock solution was added tothe tubes to produce a final SDS concentration of 0.1% (tube 3), 0.4%(tube 4), 1% (tube 5), 2% (tube 6), 10% (tube 7). Control tubes 1 and 2did not contain SDS during disruption step, with tube 2 having SDS addedafter the disruption step. The clearance between the ceramic sphere andthe centrifuge tube inner wall was about 1 mm. The resulting mixtureswere subjected to oscillatory mechanical energy using the apparatusdescribed herein applying 100 Hz and about 300×g for 20 seconds to forma solution of disrupted plant cell components. Thereafter, the tubeswere microcentrifuged at 12,000×g in a desktop microcentrifuge for 2minutes to pellet debris, and the DNA in the supernatant was isolated asdescribed in Example 6 using adsorption to silica particles.

The resulting isolated DNA was then analyzed by agarose gelelectrophoresis using equal aliquots from each sample, followed byethidium bromide staining to visualize the electrophoresed DNA, and theelectrophoresis results are shown in FIG. 14. The lanes of the gelcontain samples as follows:

Lane C Lambda Hind III DNA marker Lane 1 tube 1 (+ bead, no SDS) Lane 2tube 2 (+ bead, add 1% SDS after disruption) Lane 3 tube 3 (+ bead, 0.1%SDS) Lane 4 tube 4 (+ bead, 0.4% SDS) Lane 5 tube 5 (+ bead, 1% SDS)Lane 6 tube 6 (+ bead, 2% SDS) Lane 7 tube 7 (+ bead, 10% SDS)

The results shown in FIG. 14 demonstrate that the amount of DNA shearinginto lower molecular weight forms was inversely proportional to theamount of detergent present during the mechanical energy disruptionstep. The least amount of DNA shearing occurred in the presence of thehighest amount of SDS tested (10%), and yields appear to be the highestwith 2% SDS. Disruption of medium soft tissue using particles in theapplied energy medium in the absence of detergent results in shearing ofthe high molecular weight DNA (lanes 2-3), whereas, addition ofdetergent increases both the efficiency of DNA isolation and the qualityof isolated high molecular weight DNA.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of thepresent invention.

What is claimed is:
 1. A method of isolating high molecular weightnucleic acid from a biological material which comprises mechanicallyreleasing the high molecular weight nucleic acid from the material bythe application of rapidly oscillating reciprocal mechanical energy tothe material in the presence of a liquid medium an a closed container toproduce a released high molecular weight nucleic acid solution, whereinthe released high molecular weight nucleic acid solution has an averagemolecular weight greater than 10 kilobases, the liquid medium containsone or more particles and detergent in an amount of from 0.1% to 10%weight per weight (w/w), wherein when the liquid medium contains morethan one particle, the particles are identical or vary in size, shape,and density, and wherein the application of the energy is conducted bysubjecting the container and thereby the material to oscillations at anoscillatory rate of between about 25 hertz (Hz) to about 166 Hz for aperiod of time of between about 3 seconds to about 5 minutes, the methodfurther comprising recovering the high molecular weight nucleic acidfrom the liquid medium.
 2. The method of claim 1, wherein the particlesoccupy a volume equal to about 5% to 80% of the liquid medium volume. 3.The method of claim 1, wherein the one or more particles is onespherical bead.
 4. The method of claim 3, wherein the container hassubstantially cyclindrical walls and the spherical bead has a diameterof about 3 to 10 mm, and clearance between the spherical bead and theinner container wall is from about 0.025 to 3.0 mm.
 5. The method ofclaim 3, wherein the spherical bead occupies a volume equal to fromabout 5% to 80% of the liquid medium volume.
 6. The method of claim 1,wherein the biological material is a medium soft tissue, the oscillatoryrate is about 100 Hz producing about 300×g, and the time period is about20 to 45 seconds.
 7. The method of claim 6, wherein the medium softtissue is selected from the group consisting of heart, muscle, bloodvessels, tumor or tissue biopsies, immature plant tissue, fruit,flowers, sprouts, young leaves, nematodes, culture cells, and bacteria.8. The method of claim 1, wherein the biological material is a mediumhard tissue, the oscillatory rate is about 100 Hz producing about 300×g,and the time period is about 20 to 45 seconds.
 9. The method of claim 8,wherein the one or more particles is one spherical bead.
 10. The methodof claim 8, wherein the container has substantially cyclindrical walls,the spherical bead has a diameter of about 3 to 10 mm, and clearancebetween the spherical bead and the inner container wall is from about0.025 to 3.0 mm.
 11. The method of claim 10, wherein the clearance isabout 1 mm.
 12. The method of claim 8, wherein the medium hard tissue isselected from the group consisting of skin, cartilage, soft bone, tailsnips, ear snips, mature plant tissue such as mature leaves, stems,tubers, legumes, chitinous tissues, whole insects, slime mold, yeast,algae, viruses, and fungi.
 13. The method of claim 1, wherein thebiological material is a hard tissue, the oscillatory rate is about 100Hz producing about 300×g, the time period is about 30 to 60 seconds, andsaid container includes one or more steel spherical particles having avolume of about 1 to 50% of the liquid medium volume.
 14. The method ofclaim 13, wherein the container has substantially cyclindrical walls,the spherical bead has a diameter of about 3 to 10 mm, and clearancebetween the spherical bead and the inner container wall is from about0.025 to 3.0 mm.
 15. The method of claim 13, wherein the hard tissue isselected from the group consisting of seeds, bark, plant stems, treetrunks, rice soybean, oats, wheat, corn leaf, kernels, grains, roots,bones, soil, and fossils.
 16. The method of claim 1, wherein therecovering comprises treating the sample with an organic extractionprocedure, a chaotropic salt procedure, or an enzymatic procedure priorto isolating the high molecular weight nucleic acid.
 17. The method ofclaim 16, wherein the high molecular weight nucleic acid is isolated bylyophilization, freeze-drying, salt precipitation, or chromatographicmethods.
 18. The method of claim 1, wherein the recovering comprisesremoving non-high molecular weight nucleic acid components from theliquid medium by binding the components to a solid support.
 19. Themethod of claim 1, wherein the recovering comprises removing non-highmolecular weight nucleic acid components from the liquid medium byselectively precipitating or chromatographically removing thecomponents.
 20. The method of claim 1, wherein the recovering comprisesremoving high molecular weight nucleic acid components from the liquidmedium by selectively precipitating the high molecular weight nucleicacid from non-high molecular weight components.